The present invention relates to a feline granulocyte colony stimulating factor and a gene which codes for the factor, and, more particularly, to a feline granulocyte colony stimulating factor which is useful as a substance which functions mainly for the production of neutrophiles and maintenance of its functions in a cat, and to a method for producing the same.
It is already known that a granulocyte colony stimulating factor (G-CSF) functions mainly for the production and activation of neutrophiles which are essential for the defense in vivo. For example, human G-CSF was, for the first time, purified from xe2x80x9cCHU-2xe2x80x9d cells by Nomura et al. and its amino acid sequence was determined (Nomura et al., 1986, EMBO J., 5, p.p.871-876).
In addition, Nagata et al. carried out the cloning of its cDNA (Nagata et al., 1986, EMBO J., 5. p.575-581).
Human G-CSFs are secretory proteins which have a molecular weight of about 19 kDa. One of them consists of 204 amino acids, and contains a signal sequence which consists of 30 amino acids. Another consists of 207 amino acids. Val-Ser-Glu is inserted between Leu35 and Cys36 of the former to give the latter. As a result, the in vitro biological activity of the latter G-CSF is about one tenth of the former. It is considered that the former, which consists of 174 amino acids and is more active, mainly exists and plays important roles in vivo.
If a recombinant human G-CSF pharmaceutical preparation is administered in vivo, it stimulates the production of neutrophiles at very high yield and effectively depending on the dose and the period, enhances the number of peripheral blood neutrophiles, activates the function of the increased mature neutrophiles, and mobilize the hematopoietic stem cells to the peripheral blood. These phenomena disappear rapidly after the administration is stopped. In addition, even if a considerably large amount of it is administered, side effects are seldom observed. Even if it is administered for a long period, the stem cells are not exhausted, and antibodies are not produced. Therefore, it can be used for the supporting therapy after chemotherapy for cancer or bone marrow transplantation in order to recover quickly from neutropenia to avoid serious infectious diseases.
Recently, chemotherapy are also used for the treatment of tumors in cats. When neutrophiles decrease as in the case of human, a medicine is necessary which promotes the production of neutrophiles, but which does not have side effects. Therefore, a G-CSF, which is safe and has a strong effect, is required.
The technique to produce a large amount of a feline G-CSF by breeding cats or by culturing cells derived from a cat, has not been established yet. Purification of a feline G-CSF has not been tried, either. Therefore, it was difficult to obtain an enough amount of a feline G-CSF which is necessary for treating cats.
When dogs suffering from periodic neutropenia were treated with a human recombinant G-CSF, leukocytes increased significantly and the neutropenia was on the way to convalescence in a short period, but neutralization antibody against the human G-CSF was produced later on (Lothrop et al., 1988, Blood, 72, p.1324-1328; Hammond et al., 1991, J. Clin. Invest., 87, p.704-710).
Probably a similar phenomenon will occur in cats. It is presumably difficult to use a human or other animal""s recombinant G-CSF for treating cats for a long period.
To solve the above-mentioned problems, the inventors cloned a gene encoding a feline G-CSF to supply biologically active feline G-CSF protein inexpensively, and prepared an expression vector which has said gene. Thus the present invention was completed.
The invention according to claim 1 is directed to a protein which has I) amino acid sequence no. 2 (SEQ ID NO. 2) or II) an amino acid sequence which is obtained by deleting, substituting, adding, or inserting one or more amino acid(s) from, in, to, or into amino acid sequence no. 2 (SEQ ID NO. 2), and which has a biological activity as a feline G-CSF. The properties of amino acid sequence no. 2 (SEQ ID NO. 2) are as follows:
Sequence length: 174
Sequence type: amino acid
Topology: linear
Kind of sequence: protein
Source: cat (family Faridae, genus Felis, species catus)
Property of sequence:
characterizing symbol: feline granuloctye colony stimulating factor
Position: 1-174
Method for determining property: P
A protein which has amino acid sequence I and a biological activity as a feline G-CSF can be transcribed and translated from a gene fragment which has nucleotide sequence no. 1 (SEQ ID NO. 1) or a genomic gene which codes for a feline G-CSF, or can be produced, for example, by the chemical synthesis. Nucleotide sequence no. 1 (SEQ ID NO. 1) can be obtained by the chemical synthesis, the DNA replication, the reverse transcription, the transcription, and other methods.
A protein which has the above-mentioned amino acid sequence II which is obtained by deleting, substituting, adding, or inserting one or more amino acid(s) from, in, to, or into amino acid sequence no. 2 (SEQ ID NO. 2) and a biological activity as a feline G-CSF is encoded by a gene which is obtained by deleting, substituting, adding or inserting base(s) which correspond(s) to the above-mentioned amino acid(s) from, in, to, or into nucleotide sequence no. 1 (SEQ ID NO. 1). This protein can be transcribed and translated from this gene, or can be synthesized, for example, by a chemical method in a manner which is similar to the above-mentioned case.
According to the present invention of proteins which-have the above-mentioned amino acid sequences, a veterinary pharmaceutical composition which contains, as an active ingredient, a protein which has a biological activity as a feline G-CSF can be provided, and the composition can be effectively used as medicines for neutropenia.
In addition, a specific antibody can be obtained using the feline G-CSF protein by the well known method. This antibody can be used for immunologically detection of a G-CSF in a cat. Therefore, the said antibody can be used as a sensor for detecting and diagnosing the level of a G-CSF in a cat. In addition, the antibody can be used in the affinity chromatography for isolating and purifying said feline G-CSF protein from products by a transformant which is described below.
The present invention provides a host (transformant) which was prepared to produce said protein by recombinant techniques to express for the above-mentioned feline G-CSF gene, and a method for inexpensively producing a feline G-CSF protein using the transformant.
The above-mentioned transformant can be prepared, for example, as a xe2x80x98feline G-CSFxe2x80x99-producing cell.
In case the host cell is an eukaryotic cell, a xe2x80x98feline G-CSFxe2x80x99-producing cell can be prepared by linking a DNA fragment which has a feline G-CSF gene fragment which has nucleotide sequence no. 1 (SEQ ID NO. 1) at downstream to a promotor, such as one from viruses, followed by introducing it to eukaryotic cells. In case a recombinant virus vector such as baculovirus is used, a recombinant virus vector can be prepared, for example, according to the method described by Smith et al. (Smith et al., Mol. Cell. Biol., 3, p. 2156-2165). A transfer vector can be constructed by inserting a DNA fragment which codes for a feline G-CSF at downstream of a polyhedrin promotor of a baculovirus. The resultant transfer vector is transfected into an insect cell together with a baculovirus genome DNA. Recombinant virus clones can be selected whether they can produce G-CSF activity when infected to insect cells. In addition, a recombinant baculovirus can be prepared which produces feline G-CSF when infected to the host insects.
In case a host cell is a prokaryotic cell, a xe2x80x98feline G-CSFxe2x80x99-producing cell can be prepared, for example, by insertion of a DNA fragment which codes for a feline G-CSF protein to an expression vector, followed by transforming the prokaryotic cell.
These recombinant techniques can be carried out according to well-known, generally used protocols. Its expression efficiency can be regulated by the gene manipulation techniques and cell engineering techniques, for example, by the addition or modification of a signal sequence, the selection of a suitable host-vector system, and by the modification of an expression-regulating site of a gene. By selecting a host, the protein can also be obtained which has appropriate sugar chain(s).
The objective feline G-CSF proteins can be obtained by culturing the above-mentioned transformed cell, followed by recovering and purifying the produced proteins.
Purification of proteins from the above-mentioned cultured cells can be carried out by usual methods including the affinity chromatography, preferably the affinity chromatography which has resin conjugated with the above-mentioned specific antibody.
Therefore, by using a transformant described above, prepared according to the present invention, feline G-CSF proteins can be produced in a large amount, and can be used as medicines for neutropenia of cats.
These procedures can be carried out according to well known gene manipulation techniques and cell engineering techniques which were described, for example, by Sambrook et al. (Sambrook et al., 1989, Molecular cloning: a laboratory manual, 2nd edition, Cold Spring Harbor, N.Y.: Cold Spring Harbor Press).